PROJECT SUMMARY/ABSTRACT Multidrug resistant (MDR) Acinetobacter baumannii has emerged as a frequent cause of nosocomial infections with some isolates resistant to all clinically relevant antibiotics. We have previously identified a type VI secretion system (T6SS) in this organism. The multi-component T6SS apparatus facilitates a dynamic contact-dependent injection of toxic effector proteins into competing bacteria. The T6SS is energetically costly, and therefore in most bacteria appears to be exquisitely regulated. We recently showed that several MDR A. baumannii isolates harbor a large, self-transmissible resistance plasmid that negatively regulates T6SS. We found that T6SS is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS- mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. We propose that differentiation of A. baumannii cells into bacterial killers involves multiple phenotypic and metabolic changes and that the fitness costs associated with the MDR and T6SS phenotypes are the driving forces for this differentiation. RNAseq and differential quantitative proteomics experiments revealed that unexpected metabolic pathways related to amino acid catabolism were plasmid-regulated. Most of these metabolic changes seem to be consequence of energetic adaptations to T6SS activation and carriage of a MDR plasmid. By mutagenesis and comparative fitness assays we will determine the importance of these metabolic changes. Interfering with these pathways may result in novel strategies to combat A. baumannii infections. We will investigate how T6SS is regulated in MDR strains that do not carry plasmids to extend our conclusions to these strains. The mechanisms by which the T6SS apparatus crosses the peptidoglycan layer of the killer cell has not been determined in any bacteria. We will define the role of a putative peptidoglycanase in this process. We have also discovered phenotypic adaptations related to plasmid loss, involving piliation and motility. The biological significance of these changes will be assessed. RNAseq data led us to the hypothesis that a metabolic intermediate, phenylacetic acid (PAA), is employed as chemoattractant to recruit prey and increase the killing efficiency. Determining the role of PAA in T6SS mediated killing may result in a new paradigm for T6SS-mediated killing with important ecological implications. The outcome of this work will be a detailed understanding of the interplay between the T6SS and the MDR phenotype and the physiological changes associated to this activation, which may lead to the development of new strategies to treat Acinetobacter infections.